The field of the present invention is high sensitivity sequence analysis using radioactively labelled phenyl isothiocyanate (PITC) in the Edman degradation of peptides and proteins.
High sensitivity sequence analysis using radioactively labelled PITC has been demonstrated by a number of investigators. Bridgen, et. al., Nature, Vol. 261 (1976) 200; Howard, Nature, Vol. 261 (1976) 189; Silver and Hood, Proc. Nat. Acad, Sci. Vol. 73 (1976) 599; Bridgen, FEBS Letters Vol. 50 (1975) 159; Silver and Hood, Anal. Biochem. Vol. 67 (1975) 392; Jacobs, Niall, et. al., Fed. Proc. Vol. 32 (1973) 2445.
The principles of the Edman degradation technique for high sensitivity analysis of peptides and proteins are well-known. Briefly, the process can be described as follows. First, the protein or peptide is covalently bonded to an inert support, such as a polystyrene or glass support. While in a reaction column, this peptide or protein undergoes a coupling step, a labelling procedure using radioactive and then non-radioactive PITC. A buffer solution is pumped simultaneously with the radioactive and non-radioactive PITC, and preferably for a period of time thereafter to clean the lines. After this coupling step, the phenylthiocarbamyl peptide or protein is washed first with methanol and then with dichloroethane (DCE). The sample then undergoes a cleavage step by being treated with anhydrous trifluoroacetic acid (TFA). After the cleavage step, the protein or peptide derivatives in the reaction column are preferably washed with methanol. The resulting product after this cleavage step is an anilinothiazolinone (ATZ) amino acid derivative, which is usually unstable and normally requires a conversion step to a phenylthiohydantoin (PTH) amino acid derivative. These PTH derivatives are then identified by conventional means.
The use of .sup.35 S- or .sup.14 C- or .sup.3 H- labelled radioactive PITC provides labelled PTH amino acids that can be identified by two dimensional thin layer chromatography and autoradiography, or by isotope dilution and chromatography. While as little as 2 to 5 nanomoles of peptide or protein is sufficient for sequence determination of 15-20 residues, sequence determinations have been made on as little as picomolar quantities of protein and peptide using the above high sensitivity techniques.
The disadvantages of the prior art high sensitivity sequence determinations are numerous. First, with prior art techniques, the standard sequencer plumbing system had to be extensively modified whenever the operator wanted to introduce the radioactive PITC for labelling the protein material, a process step which is called "microsequencing" in the industry. ("Microsequencing" as used herein is intended to convey the concept of separate introduction of a radioactive reagent for labelling in the Edman degradation process. Typically, in the solid phase form in the prior art the radioactive PITC would be installed to replace the non-radioactive PITC. Then the non-radioactive PITC would be installed to replace the buffer reagent. Finally, the buffer reagent would be moved to replace the DCE reagent and the DCE reagent would be eliminated altogether. These numerous plumbing changes made the microsequencing set-up a semi-permanent situation. Interchangeability between the standard sequencer plumbing system and the microsequencing system was time consuming and hazardous to the operator because of the handling of radioactive material. Also the elimination of the DCE reagent results in lower yields for the overall sequence analysis of peptides and proteins.